PROJECT SUMMARY Monocytes are innate immune cells with diverse functions in host defense against infection and tumors, as well as tissue homeostasis, repair and regeneration. Monocytes perform surveillance roles in tissues throughout the body, and are stored in the bone marrow for rapid recruitment to infected or injured tissues. Two major classes of functionally distinct monocytes are found in the bone marrow, blood and spleen: classical (Ly6Chi in mice) and non-classical (Ly6C?) monocytes. However, additional functionally diverse monocyte subsets have recently been reported to be produced in the steady-state and under stress conditions such as infection. The molecular mechanisms underlying production of these subsets by bone marrow progenitors are poorly defined. Our studies have challenged the current, widely-accepted model of myelopoiesis and revealed 2 independent pathways of classical Ly6Chi monocyte production in the bone marrow: via granulocyte-monocyte progenitors (GMPs) and monocyte-DC progenitors (MDPs). We found that GMP- and MDP-derived Ly6Chi monocytes are morphologically distinct and exhibit differences in gene expression that are defined by their origins. GMP- derived Ly6Chi monocytes are larger and more granular than MDP-derived Ly6Chi monocytes, and enriched for granule proteins including myeloperoxidase. Thus GMP- and MDP-derived Ly6Chi monocytes appear to be functionally distinct. Moreover, we discovered that the two pathways of monocyte production are mobilized independently in response to microbial stimuli. For example, lipopolysaccharide (LPS) administration increases neutrophil and monocyte production by GMPs, while CpG DNA promotes monocyte and DC production by MDPs. Thus, the existence of two independent pathways of monocyte differentiation permits production of specific combinations of myeloid cell types during the emergency myelopoiesis response to pathogens. Our objective is to define pathways and mechanisms of monocyte production by GMPs and MDPs in the steady-state and in response to infection. In Aim 1, we will determine how myeloid transcription factors and epigenetic modifications shape the functional programming of Ly6Chi monocytes produced via the GMP and MDP pathways. In Aim 2, we will define the potential of GMP- and MDP-derived Ly6Chi monocytes to give rise to inflammatory macrophages and monocyte-derived DCs. In Aim 3, we will determine how LPS and CpG selectively target GMPs and MDPs to shape the repertoire of myeloid subsets produced. We have assembled a team of immunologists, hematologists and computational biologists to perform a set of experiments that will provide important insight into the molecular mechanisms that underlie the production of functionally diverse monocytes in the steady-state and under stress conditions. Moreover, our studies will lay the groundwork for the development of new translational approaches to specifically target functionally distinct monocyte subsets, which could be employed to promote immunity against pathogens or tumors, suppress inflammatory pathology, or facilitate tissue repair.